Abstract
Cardiovascular disease is the deadliest and most costly disease in the United States. Damaged cardiomyocytes (CMs) are not replenished via regeneration post-injury. However, human induced pluripotent stem cells (hiPSCs) can be differentiated into CMs for use in treatment and research. Homogeneous cultures of the CM subtypes are needed for effective clinical use and research. Unfortunately, current differentiation protocols lead to heterogenous cultures. To achieve homogeneous cultures, we must elucidate how growth factors and small molecules influence hiPSCs during their specification into mesoderm and, subsequently, into the distinct CM subtypes. HiPSCs can be directed into cardiac mesoderm using the growth factors bone morphogenetic protein 4 (BMP4) and Activin A or through Wnt pathway activation, and then into cardiac progenitor cells by inhibition of the Wnt pathway. Recently, the ratio of BMP4 to Activin A have been shown to direct specification of hiPSCs into pro-atrial or pro-ventricular mesoderm, but the effects of direct Wnt activation on mesoderm specification was unknown. I investigated how small molecule regulation of the Wnt and transforming growth factor beta (TGF-β) pathways affects the specification of hiPSC-derived CMs into the CM subtypes. CHIR99021 is a small molecule GSK inhibitor (GSKi) used to direct hiPSCs into mesoderm via β-Catenin activation. IWR1 is a Wnt inhibitor used to direct cardiac mesoderm into the CM subtypes. Retinoic acid (RA) when used in differentiation protocols has been shown to lead to increased atrial populations. K02288 (K) is a small molecule that inhibits the BMP signaling pathway. SB432543 (SB) is a small molecule TGFβ and Nodal inhibitor. The effects of SB on differentiation are unknown. We hypothesized that SB may inhibit Pituitary homeobox (PITx)2c in the Nodal pathway allowing for the formation of sinoatrial node like pacemaking cells. To determine the effect of Wnt activation during mesoderm induction we tested the GSKi and Activin A/BMP4 separately and in combination. In the resulting mesoderm populations, atrial marker ALDH1A2 was increased in all differentiation conditions that included GSKi. Conversely, ventricular marker CYP26A1 was down regulated in all GSKi protocols. We concluded that differentiation protocols including GSKi direct hiPSCs into pro-atrial mesoderm cells. The effect of Wnt inhibition, BMP inhibition, Nodal inhibition and RA on hiPSC differentiation were tested during the mesoderm to progenitor stage. The pro-atrial mesoderm and pro-ventricular mesoderm were treated with the small molecules IWR1, RA, SB, and K, individually and in combination to determine their effects on CM subtype specification. Protocols using SB and RA independently and in combination resulted in increased expression of atrial-specific K+ channel Kv1.5 and decreased expression of ventricular marker myosin light chain (MLC)2v. Treatments including SB and RA also increased expression of pacemaking genes short stature homeobox (SHOX)2, hyperpolarization-activated cyclic nucleotide-modulated channel (HCN)1, HCN4, Islet (ISL)1, T-box (TBX)3, and TBX18, indicating differentiation into pacemaking like cells. These results reaffirm that RA and SB are both promoting the atrial and pacemaking lineage, but SB may be more efficient in promoting pacemaker cells than RA given that the pacemaking gene expression for some pacemaking genes was higher and the anti-sinoatrial node formation Pitx2c was lower. Taken together, these data suggest that differentiation protocols including the use of small molecules CHIR99021 during mesoderm induction and SB during cardiac specification may direct hiPSCs into atrial and pacemaking like CMs.